Naturally occurring tubulin-containing paracrystals in Allogromia: immunocytochemical identification and functional significance

Bundles of microtubules (MTs) are readily visualized in vivo by videomicroscopy in highly flattened reticulopodia of the foraminiferan protozoan Allogromia sp. strain NF. In this report we use videomicroscopy, immunocytochemistry, and high-voltage electron microscopy to characterize the dynamic changes that occur in this extensive MT cytoskeleton, and in the associated cytoplasmic transport, during induced withdrawal and subsequent reextension of reticulopodia. Within seconds after application of the withdrawal stimulus (seawater substitute made hypertonic with MgCl2) intracellular bidirectional transport along linear MT-containing fibrils ceases and is replaced by an inward, constant-velocity flow of cytoplasm along the fibrils. As withdrawal continues, most fibrils become wavy and coalesce to form phase-dense pools. These wavy fibrils and phase-dense pools contain a paracrystalline material and few if any MTs. Same-section correlative immunofluorescence and high-voltage electron microscopy reveal that the paracrystalline material contains tubulin. During recovery linear fibrils (MTs) rapidly extend from the phase-dense pools (paracrystals), which concurrently shrink in size, thus reestablishing normal network morphology and motility. We conclude that the MT cytoskeleton in Allogromia reticulopodia is transformed during withdrawal into a tubulin-containing paracrystal, which serves as a temporary reservoir of MT protein and an initiation site for MT regrowth.     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

Intermediate filament-associated cytolinker plectin 1c destabilizes microtubules in keratinocytes

The transition of microtubules (MTs) from an assembled to a disassembled state plays an essential role in several cellular functions. While MT dynamics are often linked to those of actin filaments, little is known about whether intermediate filaments (IFs) have an influence on MT dynamics. We show here that plectin 1c (P1c), one of the multiple isoforms of the IF-associated cytolinker protein plectin, acts as an MT destabilizer. We found that MTs in P1c-deficient (P1c^−/−) keratinocytes are more resistant toward nocodazole-induced disassembly and display increased acetylation. In addition, live imaging of MTs in P1c^−/−, as well as in plectin-null, cells revealed decreased MT dynamics. Increased MT stability due to P1c deficiency led to changes in cell shape, increased velocity but loss of directionality of migration, smaller-sized focal adhesions, higher glucose uptake, and mitotic spindle aberrations combined with reduced growth rates of cells. On the basis of ex vivo and in vitro experimental approaches, we suggest a mechanism for MT destabilization in which isoform-specific binding of P1c to MTs antagonizes the MT-stabilizing and assembly-promoting function of MT-associated proteins through an inhibitory function exerted by plectin''s SH3 domain. Our results open new perspectives on cytolinker-coordinated IF-MT interaction and its physiological significance.     

Pb/Cu effects on the organization of microtubule cytoskeleton in interphase and mitotic cells of Allium sativum L.

The effects of lead and copper on the arrangement of microtubule (MT) cytoskeleton in root tip cells of Allium sativum L. were investigated. Batch cultures of garlic were carried out under defined conditions in the presence 10⁻⁴ M Pb/Cu of various duration treatments. With tubulin immunolabelling and transmission electron microscopy (TEM), we found four different types of MT structures depending on the cell cycle stage: the interphase array, preprophase band, mitotic spindle and phragmoplast were typical for the control cells. Pb/Cu affected the mechanisms controlling the organization of MT cytoskeleton, and induces the following aberrations in interphase and mitotic cells. (1) Pb/Cu induced the formation of atypical MT arrays in the cortical cytoplasm of the interphase cells, consisting of skewed, wavy MT bundles, MT fragments and ring-like tubulin aggregations. (2) Pb/Cu disordered the chromosome movements carried out by the mitotic spindle. The outcome was chromosome aberrations, for example, chromosome bridges and chromosome stickiness, as well as inhibition of cells from entering mitosis. (3) Depending on the time of exposure, MTs disintegrated into shorter fragments or they completely disappeared, indicating MT depolymerization. (4) Different metals had different effects on MT organization. MTs were more sensitive to the pressure of Cu ions than Pb. Moreover, TEM observations showed that the MTs were relatively short and in some places wavy when exposed to 10⁻⁴ M Pb/Cu solutions for 1–2 h. In many sections MTs were no longer visible with increasing duration of treatment (>4 h). Based on these results, we suggested that MT cytoskeleton is primarily responsible for Pb/Cu-associated toxicity and tolerance in plants.     

Novel function of eubacterial flagella: role in aggregation of a marine bacterium

Pseudomonas marina (ATCC 27 129) rapidly aggregates when suspended in buffered artificial seawater (ASW). Light microscopic observations of stained preparations, showed that flagella-flagella contact was responsible for this phenomenon. Aggregation did not occur if flagella were sheared off, or if motility was inhibited with NaN₃. Aggregates were not observed when Mg²⁺ was omitted from ASW, even though the bacteria remained motile. Other divalent cations, including Ca²⁺, Mn²⁺, and Ba²⁺ could replace Mg²⁺. However, there is no absolute requirement for divalent cations, since aggregation occurred in ASW containing Cs⁺ or Li⁺ instead of Mg²⁺. P. marina aggregates developed from pH 5.8–8.4, but not below pH 5.8 even though motility continued unimpaired to pH 4.5.Abbreviation ASW artificial seawater     

Ultrastructural basis of mitosis in the fungusNectria haematococca (sexual stage ofFusarium solani)

Summary Microtubules (MTs) in the mitotic asters of the fungusNectria haematococca (teleomorph ofFusarium solani f. sp.pisi) pull on the spindle pole bodies (SPBs) during anaphase. To elucidate the structural basis of astral forces, we conducted an ultrastructural study using primarily freeze-substitution, three-dimensional reconstruction, and computerized numerical data acquisition and analysis. The asters were composed of numerous (68–171), mostly short (<0.5 m) MTs and varied widely in total MT length (34–83 m). Both the number and total length of MTs varied up to twofold or more among asters, even between the two asters of the same mitotic apparatus (MA). Surprisingly, less than one half (38%) of the MTs in each aster were attached to the SPB. Both the number and total length of these polar MTs varied up to twofold between the two asters of the same MA. Some asters included MTs oriented back toward the opposite SPB, whereas others did not, and the number and total length of such MTs varied among asters. These results are best interpreted by assuming that astral MTs inN. haematococca have a rapid rate of turnover and exhibit dynamic instability. Any of these parameters of astral architecture could vary during mitosis and thereby give rise to the oscillations of the mitotic apparatus that occur during anaphase B by generating unequal and fluctuating forces in the two sister asters. Astral MTs were arranged asymmetrically around the astral axis, and this asymmetry could produce the lateral movements of the SPB that occur during anaphase B. An apparently extensive system of 10nm filaments occurred in these cells, and some astral MTs were associated either terminally (at the plasma membrane) or laterally with these filaments. Such associations could be involved in the development and maintenance of astral forces.Abbreviations fMT free microtubule - MA mitotic apparatus - MT microtubule - pMT polar microtubule - SPB spindle pole body     

Changes in Na+/K+-ATPase activity, unsaturated fatty acids and metallothioneins in gills of the shore crab Carcinus aestuarii after dilute seawater acclimation

We have assessed the activity of Na⁺/K⁺-ATPase, cAMP, free fatty acids (FFA) and metallothionein (MT) in the posterior gills of the brackish water shore crab Carcinus aestuarii during acclimation to 10 ppt dilute seawater (DSW). Following 3–18 days acclimation in DSW specific activity of Na⁺/K⁺-ATPase in native gill homogenates and partially purified membrane vesicles was progressively increased, from 1.7- to 3.9-fold. After short-term acclimation of crabs in DSW with added sucrose to make media isosmotic with the haemolymph the specific Na⁺/K⁺-ATPase activity in homogenates was not increased, relative to SW enzyme activity. Moreover, hyposmotic conditions led to depletion of cAMP in gills.In partially purified membrane vesicles isolated from posterior gills, fatty acids with compositions 16:0, 18:0, 18:1, 20:4 and 20:5 dominated in both SW- and DSW-acclimated Carcinus. During a year in which the metabolic activity of crabs was increased, the arachidonic/linoleic acids ratio (ARA/LA) for DSW-acclimated crabs was markedly increased relative to that in SW. Increased Na⁺ + K⁺-ATPase activity under hyposmotic stress may be modulated at least partially by the changed proportion of fatty acids in the purified membranes of posterior gills. Long-term acclimation of shore crabs to DSW resulted in a 2.6-fold increase in cytosolic metallothionein (MT) content in posterior gills over those in SW crabs. Assuming an antioxidant role of MT associated with intracellular zinc partitioning, the observed MT induction in posterior gills may be considered an adaptive response of C. aestuarii to hyposmotic stress.     

Root contraction in hyacinth III. Orientation of cortical microtubules visualized by immunofluorescence microscopy

Summary Cortical microtubules (MTs) were visualized in root cortex cells ofHyacinthus orientalis L. using immunofluorescence techniques. Cellular MT orientation was determined adjacent to radial longitudinal and transverse walls of root tip, uncontracted, contracting, and fully contracted regions. As seen in longitudinal views, MTs formed parallel, apparently helical arrays which were oriented transversely, axially or obliquely depending upon the region. Transverse sectional views showed that MTs adjacent to transverse cell walls formed a variety of patterns which varied with developmental stage and cell location. Microtubules were oriented in crisscross or parallel arrays. The parallel arrays were oriented either parallel, perpendicular or oblique to the radius of the root. There was an apparent temporal progression in MT reorientation from outer cortical to inner cortical cell layers. A resultant progression of reoriented cell growth could account for root contraction. These findings corroborate earlier electron microscopic observations of changing MT orientation accompanying root contraction, and provide cytological evidence to test mathematical and biophysical models of the mechanics of cell expansion.Abbreviations MT microtubule - MF microfibril - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.     

Variations in plant metallothioneins: the heavy metal hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> as a study case

Plant metallothioneins (MTs) are extremely diverse and are thought to be involved in metal homeostasis or detoxification. Thlaspi caerulescens is a model Zn/Cd hyperaccumulator and thus constitutes an ideal system to study the variability of these MTs. Two T. caerulescens cDNAs (accession: 665511; accession: 665515), that are highly homologous to type 1 and type 2 Arabidopsis thaliana MTs, have been isolated using a functional screen for plant cDNAs that confer Cd tolerance to yeast. However, TcMT1 has a much shorter N-terminal domain than that of A. thaliana and so lacks Cys motifs conserved through all the plant MTs classified as type 1. A systematic search in plant databases allowed the detection of MT-related sequences. Sixty-four percent fulfil the criteria for MT classification described in Cobbett and Goldsbrough (2002) and further extend our knowledge about other conserved residues that might play an important role in plant MT structure. In addition, 34% of the total MT-related sequences cannot be classified strictly as they display modifications in the conserved residues according to the current plant MTs’ classification. The significance of this variability in plant MT sequences is discussed. Functional complementation in yeast was used to assess whether these variations may alter the MTs’ function in T. caerulescens. Regulation of the expression of MTs in T. caerulescens was also investigated. TcMT1 and TcMT2 display higher expression in T. caerulescens than in A. thaliana. Moreover, their differential expression patterns in organs and in response to metal exposure, suggest that the two types of MTs may have diverse roles and functions in T. caerulescens.     

INFLUENCE OF SALINITY ON THE FORM OF ASTEROCYTIS IN PURE CULTURE1

The growth form of an isolate of Asterocytis ornata in pure culture changed from filamentous in a full-strength seawater medium to a unicellular or bicellular form closely resembling the genus Chroothece in a medium of quarter-strength seawater. The alga did not grow in media of lower salinities, or in media in which seawater was replaced by KCl, NaCl, or CaCl₂. The formation of filaments from single cells could not be attributed to osmolarity. The controlling factors seemed to be the concentrations of cations, notably Na⁺ and Mg⁺⁺. However, the filaments produced experimentally in a defined medium, were never as extensively developed as those occurring in seawater, an indication that further studies should be done. The occurrence of the Asterocytis form, in freshwater habitats remains to be explained. There may be distinct physiological races, if not true species, within the genus.     

On the interaction of ribosomal protein RPL22e with microtubules

Microtubule (MT) protein preparations often contain components of the translation machinery, including ribosome proteins. To understand the biological meaning of it we studied the interaction of ribosomal protein RPL22e with the MT. We found that bacteria expressed purified RPL22e‐GFP‐6His did co‐sediment with brain tubulin MTs with 1.3 µM dissociation coefficient. Such a K_D is comparable to some specific MT‐associated proteins. Distinct in vitro interaction of RPL22e‐GFP with MTs was also observed by TIRF microscopy. In real‐time assay, RPL22e‐GFP molecules stayed bound to MTs for several seconds, and 15% of them demonstrated random‐walk along MTs with diffusion coefficient 0.03 µ²/s. Deletion of basic areas of RPL22e did not have an impact on K_D, and deletion of acidic tail slightly increased association with MTs. Interestingly, the deletion of acidic tail increased diffusion coefficient as well. The interaction of RPL22e with MTs is hardly noticeable in vivo in cultured cells, probably since a significant part of the protein is incorporated into the ribosomes. The mobility of ribosomal protein on the MTs probably prevents its interfering with MT‐dependent transport and could ameliorate its transport to the nucleus.     

Microtubule cytoskeleton in intact and wounded coenocytic green algae

Microtubule (MT) arrangements were investigated, with immunofluorescence and electron microscopy, in two related species of coenocytic green algae. Intact cells of both Ernodesmis verticillata (Kützing) Boergesen and Boergesenia forbesii (Harvey) Feldmann have two morphologically distinct populations of MTs: a highly regular cortical array consisting of a single layer of parallel, longitudinal MTs; and perinuclear MTs radiating from the surface of the envelope of each interphase nucleus. In both algae, mitotic figures lack perinuclear MTs around them. Pre-incubation with taxol does not alter the appearance of these arrays. The cortical and nuclear MTs appear to coexist throughout the nuclear cycle, unlike the condition in most plant cells. At the cut/contracting ends of wounded Ernodesmis cells, cortical MTs exhibit bundling and marked convolution, with some curvature and slight bundling of MTs throughout the cell cortices. In Boergesenia, wound-induced reticulation and separation of the protoplasm into numerous spheres also involves a fasciation of MTs within the attenuating regions of the cytoplasm. Although some cortical MTs are fairly resistant to cold and amiprophos-methyl-induced depolymerization, the perinuclear ones are very labile, depolymerizing in 5–10 min in the cold. The MT cytoskeleton is not believed to be directly involved in wound-induced motility in these plants because amiprophos-methyl and cold depolymerize most cortical MTs without inhibiting motility. Also, the identical MT distributions in intact cells of these two algae belie the very different patterns of cytoplasmic motility. Although certain roles of the MT arrays may be ruled out, their exact functions in these plants are not known.Abbreviations APM amiprophos-methyl - DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MT(s) microtubule(s) - PBS phosphate-buffered saline     

Arabidopsis microtubule-associated protein AtMAP65-2 acts as a microtubule stabilizer

Nine genes that encode proteins of the MAP65 family have been identified in the Arabidopsis thaliana genome. In this study, we reported that AtMAP65-2, a member of the AtMAP65 family, could strongly stabilize microtubules (MTs). Bacterially-expressed AtMAP65-2 fusion proteins induced the formation of large MT bundles in vitro. Although AtMAP65-2 showed little effect on MT assembly or nucleation, AtMAP65-2 greatly stabilized MTs that were subjected to low-temperature treatment in vitro. Analyses of truncated versions of AtMAP65-2 indicated that the region that encompassed amino acids 495–578, which formed a flexible extended loop, played a crucial role in the stabilization of MTs. Analysis of suspension-cultured Arabidopsis cells that expressed the AtMAP65-2-GFP fusion protein showed that AtMAP65-2 co-localized with MTs throughout the cell cycle. Cortical MTs that were decorated with AtMAP65-2-GFP were more resistant to the MT-disrupting drug propyzamide and to ice treatment in vivo. The results of this study demonstrate that AtMAP65-2 strongly stabilizes MTs and is involved in the regulation of MT organization and dynamics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. H. Li and X. Zeng have contributed equally to this paper and are considered as joint first authors.     

Re-formation of microtubules in Closterium ehrenbergii Meneghini after cold-induced depolymerization

Immunofluorescence microscopy was used to examine the re-formation of microtubules (MT), after cold-induced depolymerization, in Closterium ehrenbergii. The C. ehrenbergii cells undergo cell division followed by semicell expansion in the dark period of daily light-dark cycles. Five types of MTs, namely the MT ring, hair-like MTs around the nuclei, spindle MTs, radially arranged MTs and transverse wall MTs, appeared and disappeared sequentially during and following cell division. The wall MTs were distributed transversely only in the expanding new semicells. When cells were chilled in ice water, wall MTs in expanding cells were fragmented, and then disappeared as did the other types of MTs, within 5 min. When cells were warmed at 20°C after 2 h chilling, wall MTs and the other types of MTs re-formed. At the early stage of wall-MT re-formation in expanding cells, small, star-like MTs were formed, and then randomly oriented MTs developed in both the expanding new and the old semicells. The MT ring was also re-formed at the boundary between the new and old semicells. There were no obvious MT-organizing centers in the random arrangement. As time passed, the randomly oriented wall MTs in the old semicells disappeared and those in the expanding new semicells gradually assumed a transverse orientation. These results indicate that wall MTs can be rearranged transversely after they have been re-formed and that nucleation of wall MTs is separable from the mechanism for ordering them.Abbreviations MT(s) microtubule(s) - MTOC(s) microtubule-organizing center(s)     

Studies on the motility of the foraminifera. I. Ultrastructure of the reticulopodial network of Allogromia laticollaris (Arnold)

Allogromia laticollaris, a benthic marine foraminifer, extends numerous trunk filopodia that repeatedly branch, anastomose, and fuse again to form the reticulopodial network (RPN), within which an incessant streaming of cytoplasmic particles occurs. The motion of the particles is saltatory and bidirectional, even in the thinnest filopodia detected by optical microscopy. Fibrils are visible by differential interference microscopy, and the PRN displays positive birefringence in polarized light. These fibrils remain intact after lysis and extraction of the RPN in solutions that stabilize microtubules (MTs). Electron micrographs of thin sections through these lysed and stabilized cytoskeletal models reveal bundles of MTs. The RPNs of living Allogromia may be preserved by standard EM fixatives only after acclimatization to calcium-free seawater, in which the streaming is normal. The MTs in the RPN are typically arranged in bundles that generally lie parallel to the long axis of the trunk and branch filopodia. Stereo electron micrographs of whole-mount, fixed, and critical-point-dried organisms show that the complex pattern of MT deployment reflects the pattern of particle motion in both flattened and highly branched portions of the RPN. Cytoplasmic particles, some of which have a fuzzy coat, are closely associated with, and preferentially oriented along, either single MTs or MT bundles. Thin filaments (approximately 5 nm) are also observed within the network, lying parallel to and interdigitating with the MTs, and in flattened terminal areas of the filopodia. These filaments do not bind skeletal muscle myosin S1 under conditions that heavily decorate actin filaments in controls (human blood platelets), and are approximately 20% too thin to be identified ultrastructurally as F-actin.     

Reorientation of microtubules at the outer epidermal wall of maize coleoptiles by phytochrome,blue-light photoreceptor,and auxin

Summary The effects of red and blue light on the orientation of cortical microtubules (MTs) underneath the outer epidermal wall of maize (Zea mays L.) coleoptiles were investigated with immunofluorescent techniques. The epidermal cells of dark-grown coleoptiles demonstrated an irregular pattern of regions of parallel MTs with a random distribution of orientations. This pattern could be changed into a uniformly transverse MT alignment with respect to the long cell axis by 1 h of irradiation with red light. This response was transient as the MTs spontaneously shifted into a longitudinal orientation after 1–2 h of continued irradiation. Induction/reversion experiments with short red and far-red light pulses demonstrated the involvement of phytochrome in this response. In contrast to red light, irradiation with blue light induced a stable longitudinal MT alignment which was established within 10 min. The blue-light response could not be affected by subsequent irradiations with red or far-red light indicating the involvement of a separate blue-light photoreceptor which antagonizes the effect of phytochrome. In mixed light treatments with red and blue light, the blue-light photoreceptor always dominated over phytochrome which exhibited an apparently less stable influence on MT orientation. Long-term irradiations with red or blue light up to 6 h did not reveal any rhythmic changes of MT orientation that could be related to the rhythmicity of helicoidal cell-wall structure. Subapical segments isolated from dark-grown coleoptiles maintained a longitudinal MT arrangement even in red light indicating that the responsiveness to phytochrome was lost upon isolation. Conversely auxin induced a transverse MT arrangement in isolated segments even in blue light, indicating that the responsiveness to blue-light photoreceptor was eliminated by the hormone. These complex interactions are discussed in the context of current hypotheses on the functional significance of MT reorientations for cell development.Abbreviations MT cortical microtubule - P_r, P_fr red and far-red absorbing form of phytochrome     

Microtubule-associated proteins in higher plants

A variety of microtubule-associated proteins (MAPs) have been reported in higher plants. Microtubule (MT) polymerization starts from the γ-tubulin complex (γTuC), a component of the MT nucleation site. MAP200/MOR1 and katanin regulate the length of the MT by promoting the dynamic instability of MTs and cutting MTs, respectively. In construction of different MT structures, MTs are bundled or are associated with other components—actin filaments, the plasma membrane, and organelles. The MAP65 family and some of kinesin family are important in bundling MTs. MT plus-end-tracking proteins (+TIPs) including end-binding protein 1 (EB1), Arabidopsis thaliana kinesin 5 (ATK5), and SPIRAL 1 (SPR1) localize to the plus end of MTs. It has been suggested that +TIPs are involved in binding of MT to other structures. Phospholipase D (PLD) is a possible candidate responsible for binding of MTs to the plasma membrane. Many candidates have been reported as actin-binding MAPs, for example calponin-homology domain (KCH) family kinesin, kinesin-like calmodulin-binding protein (KCBP), and MAP190. RNA distribution and translation depends on MT structures, and several RNA-related MAPs have been reported. This article gives an overview of predicted roles of these MAPs in higher plants.     

Cytoskeletal changes during resumption of tip growth in nongrowing protonemal cells of the fernAdiantum capillus-veneris L.

Summary Nongrowing, two-celled protonemata of the fernAdiantum capillus-veneris L. resume tip growth within the apical cell upon irradiation with red light. In this study, the phenomenon of growth resumption was analyzed with reference to changes in cytoskeletal organization. Continuous observations of apical cells with time lapse video-microscopy revealed that the nucleus migrated toward the tip ca. 1.9 h after the onset of red light, much earlier than the initiation of tip growth, which took place ca. 8.5 h after irradiation. Cytoskeletal organization was observed at various time points during growth resumption by fluorescent staining of microfilaments (MFs) and microtubules (MTs) with rhodamine-phalloidin and anti-tubulin antibodies. At 2 h after red-light irradiation, endoplasmic MF and MT strands appeared at the apical end of nucleus. These strands extended into the apical endoplasm, where filaments were rare prior to irradiation. Many fine filaments branched from the strands to the cell periphery, including the cortex of the apical-dome region. At this time, cortical circular arrays of MTs and MFs, normally found in the growing apex of protonemal cells, were absent. Both MT and MF circular arrays appeared during the resumption of tip growth concomitantly. The half-maximum appearance of MT and MF circular arrays within a population occurred at 5.4 h and 5.8 h after red-light irradiation, respectively. Thus, the process of red-light-induced resumption of tip growth in fern protonemal cell is composed of a series of events. These events include: (1) the appearance of strands extending from the nucleus toward the apical cortex and the migration of nucleus toward the apex; (2) the formation of circular MT and MF arrays at the sub-apical cortex; and (3) the initiation of cell growth at the apex. These results reflect the significant roles of MF and MT cytoskeleton in the resumption of tip growth.Abbreviations MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - MF microfilament - MT microtubule